The long term objective of this project is to study the mechanisms of DNA repair in a model eukaryotic cell, Chlamydomonas reinhardi. Photoreactivating enzyme disappears from nuclei of ultraviolet light-irradiated cells. The cells will be reacted with an alkylating agent, methyl methanesulfonate to determine the specificity of the damage that will trigger the disappearance of photoreactivating enzyme. A photoreactivation defective mutant will be isolated in order to determine if nuclear and chloroplast photoreactivating enzyme are controlled by different genes. The repair of alkylated bases in DNA will be studied in both wild-type and sensitive mutants. The methods used will employ chemical and enzymatic degradation followed by alkaline sucrose gradients as well as direct isolation of the alkylated bases. Partcular emphasis will be on the question of the enzymatic repair of 7-methylguanine. The dark repair of pyrimidine dimers during the various stages of germination of the diploid zygospore of Chlamydomonas will be studied. A UV-specific endonuclease and alkaline sucrose gradients will be used for the quantitation of the pyrmidine dimers.